ABSTRACT Portal fibrosis develops in chronic liver disease in which the initial primary insult is centrilobular, but the mechanism involved still remains unclear. Osteopontin (OPN) is a cytokine constitutively expressed in cells within the periportal region and it is highly induced in liver injury. We believe that OPN enables cells to sense molecular patterns associated with liver injury, and triggers signals that are required for oval cell expansion, ductular reaction, and fibrogenesis to occur. In this Competitive Renewal we will focus on testing the Central Hypothesis Oxidative stress-mediated liver injury will up-regulate OPN, which in turn, will induce oval cell expansion, ductular reaction, and collagen I expression, contributing to the development of liver fibrosis. Specifically, we hypothesize that: 1) Oxidative stress-mediated liver injury will induce OPN expression; 2) OPN will up-regulate collagen I in stellate cells acting as a feed-forward mechanism to promote scarring; 3) OPN will drive oval cell expansion and ductular reaction, and 4) Opn-/- mice will avert liver fibrosis by decreasing oval cell expansion, ductular reaction, and limiting collagen I deposition in vivo. Four Specific Aims are proposed to address these hypotheses. In Aim 1, we will evaluate in vitro the molecular basis whereby reactive oxygen species signaling up-regulates OPN in oval cells, biliary epithelial cells, and hepatic stellate cells in thioacetamide-induced liver fibrosis. In Aim 2, to study the effects of OPN on collagen I up-regulation in stellate cells, we will identify the membrane proteins engaged by OPN and the proximal signaling molecules/stress-sensitive kinases activated upon binding that trigger the pro-fibrogenic cascade. In Aim 3, to dissect the role of OPN in oval cell expansion and ductular reaction, primary oval cells will be treated with recombinant OPN and oval cell proliferation and differentiation to biliary epithelial cells, as well as the potential factors involved that may also impact on the stellate cell fibrogenic response, will be evaluated. In Aim 4, the in vivo physiological contribution of OPN induction to oval cell proliferation, ductular reaction, and the fibrogenic response will be tested in time-course experiments using wild-type and Opn-/- mice and two well-established models of liver fibrosis. Biochemical and immunohistological parameters of oval cell expansion, ductular reaction, inflammation, necrosis, hepatocyte replicative arrest, and fibrogenesis will be evaluated as read-outs for the contribution of OPN to liver fibrosis. Public Health Relevance: Liver fibrosis affects several million people in the U.S. and progresses to cirrhosis and hepatocellular carcinoma in many patients. The Goal of this Proposal is to investigate the role of OPN signaling in this process, and to evaluate whether targeting the OPNregulated network may be a useful strategy for preventing or treating liver fibrosis.